One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation

Summary To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRα and TCRβ chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8+ T cells followed by electroporation of T cells with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and single-guide RNAs targeting the TCRα and TCRβ constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.


SUMMARY
To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRa and TCRb chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8 + T cells followed by electroporation of T cells with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and single-guide RNAs targeting the TCRa and TCRb constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.

BEFORE YOU BEGIN
The CRISPR-Cas9 system has revolutionized the field of molecular biology as a versatile genome-editing tool with a broad range of applications. Guided by a short RNA molecule, Cas9 is targeted to a genomic locus and creates a double-strand break. Upon cleavage by Cas9, the targeted locus is repaired by the dominant non-homologous end joining (NHEJ) pathway. This error-prone repair mechanism re-ligates the double-strand brakes, introducing small insertions or deletions (indels) at the breakpoint. Indels within a coding exon of the gene can lead to frameshift mutations and premature stop codons, resulting in a knockout of the gene. 1 Although the CRISPR-Cas9 system is more accurate and efficient than other genome editing methods, integrating and stable delivery systems to introduce Cas9, for example those based on viral vectors, raise concerns about persistent Cas9 expression that could lead to off-target editing. Thus, non-viral methods that involve transient expression of Cas9, such as those using Cas9 ribonucleoproteins (RNPs) or Cas9 mRNA, benefit from a better safety profile. Compared to Cas9 RNPs, in-house production of Cas9 mRNA using plasmid vectors usually requires less resources and more accessible infrastructure in the context of clinical translation, making mRNA electroporation a desirable method for non-viral T-cell engineering. 2 This protocol describes an optimized single electroporation fully RNA-based CRISPR-Cas9 strategy to eliminate the native TCR genes. Purified CD8 + T cells are activated with anti-CD3 and anti-CD28 antibodies for three days. In vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA is co-electroporated with single guide RNAs (sgRNAs) specific for human TCR a-chain constant (TRAC) and TCR b-chain constant (TRBC) in activated T cells to create double-strand breaks in the native TCR loci. The resulting TCR knockout can prevent TCR mispairing between native and Institutional permissions All procedures involving human blood samples should be performed in accordance with relevant institutional and governmental ethics regulations. Informed consent should be obtained from all subjects for the experimental use of human blood. In this case, experimental work was compliant with the local Ethics Committee of the Antwerp University Hospital-University of Antwerp (Antwerp, Belgium) under project ID 0511. Selection of blood donors and collection of blood was performed according to Belgian law and Belgian Red Cross policy. This includes the signing of an informed consent in which the donor agrees that his/her blood can be used for purposes other than blood transfusion, including experimental research.

Peripheral blood mononuclear cells (PBMC) isolation from buffy coats
Timing: 3 h This step describes how to isolate PBMC from (healthy) donor buffy coats. Buffy coats primarily contain leukocytes and platelets and are derived from whole blood donations of anonymous volunteers. In this protocol, donor buffy coats were provided by the Blood Service of the Donor Center Mechelen (Red Cross-Flanders, Mechelen, Belgium). Density gradient centrifugation is used to separate the mononuclear leukocytes from the rest fraction of red blood cells (RBC), polymorphonuclear leukocytes (granulocytes) and platelets. The protocol below applies Ficoll-Paque PLUS as density gradient medium and has an estimated yield of 6.5 3 10 8 PBMCs per 40 mL buffy coat. Alternatively, other density gradient media can be used to isolate PBMC, such as Lymphoprep (Stemcell Technologies).
1. Prepare phosphate-buffered saline (PBS)/Ethylenediaminetetraacetic acid (EDTA) buffer (see materials and equipment) and pre-heat to 37 C. 2. Wipe the blood tube of the buffy coat bag with 70% ethanol solution and cut the tube to release the blood.
CRITICAL: For this point on, sterile working is important to avoid contamination of the cells.
3. Divide the buffy coat over sterile 50 mL conical centrifuge tubes ($10 mL blood per tube). 4. Dilute the blood 1:3 by adding pre-heated PBS/EDTA buffer to each tube. For 10 mL of blood, add to a final volume of 30 mL. Add the PBS/EDTA buffer fast enough to ensure good mixing.
Note: When using whole blood as starting material instead of buffy coat preparation, 1:2 dilution is recommended.
5. Carefully layer 12 mL of Ficoll-Paque PLUS under the diluted buffy coat.
CRITICAL: Mixing of Ficoll-Paque PLUS and diluted blood must be avoided to obtain a high purity of PBMC.
Note: Less Ficoll-Paque PLUS can be used but it may hinder the separation between the PBMC layer and the RBC fraction.
6. Centrifuge the tubes at 740 3 g for 30 min (min) at 19 C-22 C in a swinging bucket rotor.

OPEN ACCESS
CRITICAL: For Ficoll-layered blood samples, lower the acceleration speed of the centrifuge and inactivate the rotor brakes to effectively prevent mixing of the different phases obtained after centrifugation.
Note: After density gradient centrifugation each tube contains four layers. The top layer consists of plasma and platelets, followed by an opaque interphase with the PBMC, next the transparent Ficoll layer, and the RBC fraction at the bottom of the tube.
7. Collect the PBMC layer. a. Remove two thirds of the upper plasma layer, leaving a small volume of plasma on the PBMC interphase. b. Carefully loosen up the PBMC sticking to the side of the tube with the tip of your 10 mL pipette. c. Aspirate the PBMC layer and transfer to a clean, sterile 50 mL Falcon tube. Discard the remaining Ficoll and RBC layer. d. Repeat these steps for each tube. 8. Add PBS/EDTA to a final volume of 40 mL and centrifuge the tubes at 480 3 g for 5 min at 19 C-22 C. Centrifugation can be performed with maximal acceleration and the rotor brakes on. 9. Discard the supernatant. Pool the cells to 2 3 50 mL tubes by resuspending the pellets in 10 mL PBS/EDTA. 10. Repeat washing step 8. 11. Discard the supernatant and pool the cells to 1 3 50 mL tube in a final volume of 50 mL PBS/ EDTA. 12. After checking cell concentration with an automatic cell counter, transfer 100,000 PBMC to a polystyrene FACS tube to determine viability. a. Add 100 mL FACS buffer to the cells. Stain cells with 0.5 mL propidium iodide (PI; 1 mg/mL) and incubate for 1 min at 19 C-22 C. b. Measure cell viability on a flow cytometer with a filter set compatible with PI. A viability of 95%-99% is expected after PBMC isolation.
Note: RBC contamination is a common issue with PBMC isolation and can cause potential errors in calculation of the cell concentration and downstream primary CD8 + T-cell isolation. RBC lysis buffer can be used to eliminate the RBC from the PBMC.
Pause point: Isolated PBMC can be kept for 16 h on a roller shaker in serum-free AIM-V medium at 1-1.6 3 10 7 cells/mL or can be cryopreserved at 0.5-1 3 10 8 cells/mL in 90% FBS + 10% dimethyl sulfoxide (DMSO) stored in a À80 C freezer for short-term storage or below À150 C for long-term storage.
Positive magnetic-activated cell sorting (MACS) of primary resting CD8 + T cells

Timing: 3 h
The following section describes the immunomagnetic selection of primary resting CD8 + T cells from isolated human PBMC with human CD8 magnetic microbeads, according to the manufacturer instructions (Miltenyi). Generally, cytotoxic CD8 + T cells comprise 5%-25% of PBMC. With immunomagnetic selection, CD8 + T cells can be isolated from the unwanted cells with an expected purity of more than 95%.
Note: Alternatively to positive selection, negative selection kits (e.g., Miltenyi or STEMCell) can be used to isolate CD8 + T cells.
13. Determine the percentage CD3 + CD8 + T cells present in the PBMC. i. Acquire the stained cells on a flow cytometer and determine the percentage of CD3 + CD8 + T cells ( Figure 1A). 14. Calculate the amount of PBMC to be used for the desired number of CD8 + T cells to be isolated.
Transfer the PBMC to a clean, sterile 50 mL Falcon tube. 15. Centrifuge cell suspension at 300 3 g for 10 min at 19 C-22 C. Remove supernatant completely. 16. Follow instructions on the manufacturer's protocol.
CRITICAL: From this step on, work, fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
CRITICAL: When flushing the cells of the LS column, extra caution is required when removing the LS column from the magnetic MACS separator and placing it into a 15 mL tube to avoid contamination due to manual handling.
17. Determine the yield and purity of the isolated CD8 + T cells.
a. Take 100 mL isolated CD8 + T cells and divide over two polystyrene FACS tubes (50,000 cells/ tube). Use one tube to determine the cell concentration on an automatic cell counter and to check cell viability with PI (see step 12). A viability ranging from 90%-99% can be expected. Use the other tube to check the purity of the isolation (% of CD3 + CD8 + T cells). b. Wash the samples by adding 2 mL of FACS buffer. c. Centrifuge cells at 480 3 g for 5 min at 19 C-22 C. d. Discard the supernatant.  Pause point: Isolated CD8 + T cells can be cryopreserved at 10-50 3 10 6 in 1 mL of 90% FBS + 10% DMSO stored in a À80 C freezer for short-term or below À150 C for long-term storage. It is not recommended to cryopreserve CD8 + T cells when starting from cryopreserved PBMC.  In order to delete native TRAC and TRBC genes in CD8 + T cells with a high degree of efficiency, a rationally engineered version of Streptococcus pyogenes Cas9 (SpCas9) with enhanced specificity (eSp-Cas9(1.1)) is used. 3 The eSpCas9(1.1) sequence is inserted into a pST1 plasmid vector back-bone. 4,5 This vector has been optimized for the in vitro transcription (IVT) of mRNA to rapidly and efficiently produce high amounts of synthetic mRNA. The open reading frame of the eSpCas9(1.1) is preceded by a T7 promoter, a 5 0 cloning site, a Kozak sequence at the translational start site, and one copy of the monopartite nuclear localization signal of the simian virus 40 (SV40) T-antigen (PKKKRKV) followed by a 32 amino-acid linker. 6 The eSpCas9(1.1) is followed by a copy of the bipartite NLS from nucleoplasmin, a GSG linker, a 2A peptide from a porcine teschovirus-1 (P2A) and an enhanced green fluorescence protein (EGFP) reporter gene and the 3 0 cloning site. The P2A sequence allows co-expression of both eSp-Cas9(1.1) and EGFP by self-cleavage of the P2A peptide during translation. 7 An EGFP reporter gene is added to the construct to determine the transfection efficiency of the mRNA construct after electroporation and can be easily omitted or replaced by a preferred reporter gene. The SV40 NLS-Linker-eSpCas9(1.1)-Nucleoplasmin NLS-GSG-P2A-EGFP (in short, eSpCas9(1.1)-P2A-EGFP) insert has a total length of 5070 nucleotides and was codon optimized for Homo sapiens. The 3 0 untranslated region (UTR) present in the pST1 backbone is comprised of two copies of the 3 0 UTR from the human alpha globin gene and a 120-nucleotide poly(A) tail, which increase the stability and translational efficiency of the produced mRNA, 4 and a restriction site for a Type IIS restriction enzyme ( Figure 2).

KEY RESOURCES
1. Design the eSpCas9(1.1)-P2A-EGFP DNA construct plasmid in silico by combining the segments described above in a plasmid vector of preference. 2. Construct synthesis, cloning and plasmid preparation can be performed in-house or outsourced to commercial providers such as Gene-Art.
Optional: Any plasmid vector suitable for IVT of mRNA can be used as an alternative for the pST1 plasmid vector.  5. Incubate the microcentrifuge tube on a thermoblock for 2 h at 37 C. 6. Examine the linearized DNA template in a 1% agarose gel electrophoresis to confirm cleavage of the plasmid is complete (Figure 3).
Note: linearized plasmid DNA is precipitated with sodium acetate and ethanol solutions. Ethanol precipitation is a commonly used method to de-salt and concentrate DNA. Nucleic acids are negatively charged due to the presence of phosphate groups, making DNA readily soluble in water. Addition of sodium acetate and ethanol disrupts the hydrate shell and neutralizes the negative charge of DNA, leading to precipitation.
7. Add 50 mL of 3 M sodium acetate (pH 5.2) to the microcentrifuge tube. This is 1/10 of the final volume used to linearize 50 mg of plasmid DNA. 8. Add 1 mL of absolute ethanol to the microcentrifuge tube. This is 2 volumes of the final volume used to linearize 50 mg of plasmid DNA. 9. Mix the solution gently by pipetting. 10. Incubate 16-24 h at À20 C. 11. Centrifuge the microcentrifuge tube at 16,100 3 g for 15 min at 4 C. 12. Remove the supernatant carefully without disturbing the DNA pellet. 13. Add 1 mL of 70% ethanol solution to the DNA pellet. Do not resuspend the pellet.
Note: Place the microcentrifuge tube in the same direction in the centrifuge for each centrifugation step in order to know where the DNA pellet is, since sometimes it is difficult to see.
14. Centrifuge the microcentrifuge tube at 16,100 3 g for 15 min at 4 C. 15. Remove supernatant carefully without disturbing the DNA pellet. 16. Add 50 mL of nuclease-free distilled water to dissolve the DNA pellet.
Caution: Do not pipette up and down the DNA pellet. Instead, leave it for 30 min or longer at 4 C to dissolve. bring the linearized plasmid DNA to a concentration of 0.5 mg/mL. Approximately 90% of the starting material is recovered after linearization and precipitation.
Pause point: Linear DNA can be directly used for IVT of mRNA or stored at À20 C for at least 1 year.

Timing: 7 h
The eSpCas9(1.1)-P2A-EGFP mRNA is synthesized by IVT using the mMESSAGE mMACHINE T7 transcription kit. The protocol described below is adapted from the manufacturer's recommendations (Thermo Fisher Scientific) and uses a T7 enzyme mix containing RNA polymerases to produce large amounts of capped RNA. According to the manufacturer, this kit produces an average yield of 20-30 mg mRNA per 1 mg of linearized plasmid template and the reaction setup showed here is compatible with mRNA of 300 bases to 5 kb in length.

Thaw the frozen agents of the mMESSAGE mMACHINE T7 transcription kit.
Note: Keep the RNA Polymerase Enzyme Mix and 23 NTP/CAP on ice and keep the 103 Reaction buffer at 19 C-22 C while assembling the reaction.
CRITICAL: During the next steps it is important to work fast and use RNase-free filter pipette tips to avoid degradation of the RNAs.
19. Vortex the 103 Reaction buffer and 23 NTP/CAP. 20. Briefly spin all reagents to prevent contamination or loss of reagent. 21. Assemble the transcription reaction in a sterile 1.5 mL microcentrifuge tube at 19 C-22 C in the following order: Note: The  Optional: As quality control, IVT mRNA can be examined in a non-denaturing 1% agarose gel electrophoresis to confirm the integrity and the length of the produced mRNA ( Figure 4).

Timing: 3 h
The section below describes an in vitro activation protocol for primary CD8 + T cells via stimulation of the TCR-CD3 complex using plate-bound anti-CD3 and soluble anti-CD28 monoclonal antibodies and stimulatory cytokines. Short-term activation of T cells will enable greater RNA transfection efficiency compared to non-activated T cells and thus subsequent disruption of the native TRAC and TRBC sequences.
Note: Both MACS-isolated fresh and cryopreserved human CD8 + T cells can be used. When using cryopreserved cells, thaw them in CTL medium and let the cells rest for at least 4 h before activation (problem 3). Note: Volumes are adjusted for 20 3 10 6 primary CD8 + T cells at the beginning of the culture. Up to 30 3 10 6 primary CD8 + T cells can be cultured in a T75 cell culture flask; in that case, adjust the volumes of NA/LE anti-CD28 antibody and cytokines accordingly.

Generation of TKO8 cells with RNA electroporation
Timing: 2 h mRNA electroporation is a powerful tool for transient genetic modification of cells. 2 Combining the CRISPR gene editing tool with mRNA delivery provides a safer modification strategy compared to integrating delivery systems, for example those based on viral vectors. 8 In this step, activated primary CD8 + T cells are electroporated with eSpCas9(1.1)-P2A-EGFP mRNA and two sgRNAs targeting exon 1 of TRAC and exon 1 of TRBC to disrupt the expression of native TCRs (referred to as TKO8 cells). After transfection into CD8 + T cells, translation of eSpCas9(1.1)-P2A-EGFP mRNA will result in transient eSpCas9(1.1) expression together with EGFP. Cas9 protein will then bind to the sgRNAs, which will be transported to the nucleus, and will be guided to the TRAC and TRBC sequences of interest by the sgRNAs; with this construct, EGFP operates as a reporter for transfection efficiency.
48. Prepare EP recovery medium and pre-heat 5 mL in a 15 mL tube in a 37 C water bath. 49. Carefully resuspend activated CD8 + T cells from the T75 flask by pipetting against the bottom of the flask. 50. Take 100 mL of activated cells to determine the cell concentration on an automatic cell counter and to check cell viability with PI (see step 12, before you begin). A viability ranging from 90%-99% can be expected. 51. Collect 10 7 viable activated CD8 + T cells.
Optional: This protocol can be upscaled up to 5 3 10 7 cells in a maximum of 400 mL of electroporation buffer for a 4 mm electroporation cuvette.  82. Acquire the stained cells on a flow cytometer and determine the percentage of viable CD3 + CD8 + TCR + T cells. A TCR knockout efficiency ranging from 90% to 99% can be expected ( Figure 6) (problem 5).
Note: Native TCR knockout efficiency could alternatively be assessed on a transcriptomic level using reverse transcription quantitative real-time PCR (RT-qPCR) or on a genomic level using DNA sequencing.

EXPECTED OUTCOMES
Viral methods to introduce Cas9 raise concerns about persistent Cas9 expression that could lead to off-target editing. Non-viral methods that involve transient expression of Cas9, such as those using Cas9 ribonucleoproteins or Cas9 messenger RNA (mRNA), benefit from a better safety profile. Compared to Cas9 ribonucleoproteins, in-house production of Cas9 mRNA using plasmid vectors usually requires less resources and more easily accessible infrastructure, making mRNA electroporation a desirable method for non-viral T-cell engineering. Here, IVT of eSpCas9(1.1)-P2A-EGFP mRNA with the mMESSAGE mMACHINE T7 transcription kit generally yields G20 mg mRNA per 1 mg of linearized plasmid used for the reaction. This produced eSpCas9(1.1)-P2A-EGFP mRNA is then co-electroporated with sgRNAs specific for TRAC and TRBC in activated CD8 + T cells. With the described one-week single-electroporation RNA-based CRISPR-Cas9 protocol, TCR KO efficiency is expected to range from 90% to 99% 72 h after electroporation. Representative flow cytometric results after TCR KO are shown in Figure 6. 72 h after electroporation, native TCR expression is completely eliminated and TKO8 cells can be engineered to validate transgenic TCRs without chance of mispairing with the native TCR chains. In addition, this protocol could be applied in allogeneic T-cell therapy by non-viral and stable elimination of the native TCR to avoid GvHD. In summary, this RNA-based CRISPR/Cas9 strategy provides a robust and non-viral approach for rapid multiplex genome engineering of primary T cells.

LIMITATIONS
This protocol provides an RNA-based method to efficiently disrupt native TCR expression in primary human CD8 + T cells. One important advantage of this protocol over those based on RNPs is that fully RNA-based methods are usually more affordable when translating to the clinic, 2 and mRNA can be synthesized in-house with minimal equipment. This methodology has been optimized for the specific TRAC and TRBC sgRNA sequences described in the protocol. Alternative sgRNAs with different target sequences (either within TRAC and TRBC or within other loci) must be tested and/or optimized to achieve the greatest knockout efficiency possible. Moreover, it may be necessary to optimize the electroporation settings when using different electroporation systems as the one described here.

Problem 1
Low purity or yield of CD8 + T cells after magnetic-activated cell sorting (related to step 17 before you begin).

Potential solution
One of the possible causes of low yield of CD8 + T cells is an insufficient amount of beads added to the PBMC before magnetic-activated cell sorting. To avoid this, correctly calculate the volume of beads needed and resuspend or vortex the beads prior to adding to the cell suspension. When added to the cell suspension, mix well. A possible cause of low purity could be the obstruction of the LS column by bubbles. Avoid the formation of bubbles when transferring the labeled cells to the LS column. Lastly, the presence of RBC or dead cells can also affect the purity of the isolated CD8 + T cells. If the PBMC sample contains RBC, consider treating the PBMC sample with RBC lysis buffer. The presence of cell aggregates due to cellular debris can be avoided by adding DNase-I (50 UI/mL) to the PBMC sample.

Problem 2
Low yield of mRNA after IVT (related to step 34).

Potential solution
One of the main reasons of low mRNA yield after in vitro synthesis is degradation by RNase. Although a RNase inhibitor is present in the Enzyme Mix of the mMESSAGE mMACHINE T7 transcription kit, it can only inactivate trace RNase contaminations. Therefore, production of IVT mRNA demands a dedicated RNA bench cleaned to remove the RNases and RNase free pipettes. In addition, the DNA template used for IVT can be contaminated with residual RNase A from the miniprep or introduced RNase from restriction enzymes. The resulting mRNA appears degraded and ll OPEN ACCESS forms a smear on the quality control gel electrophoresis. In this case, proteinase K treatment of the DNA template to remove residual RNase before IVT is recommended. Lastly, a low yield can be caused by the degradation of the template DNA itself. Correct handling and storage of the linearized plasmid DNA is important.

Problem 3
Low viability and yield after thawing of CD8 + T cells (related to step 41).

Potential solution
When thawing cryopreserved cells, a fast-thawing method is important because of the toxic effect of DMSO at room temperature on cells. Adding thawed cells to an excess of prewarmed CTL medium dilutes DMSO to a non-toxic concentration. By centrifugation at 480 3 g for 5 min at 19 C-22 C and resuspension in new CTL medium, DMSO is washed away. Additionally, to avoid more loss of T cells ensure that cryopreserved cells are recovered from the cryopreservation. Therefore, wait at least 4 h prior to culturing T cells.

Problem 4
Loss of CD8 + T cells after activation (related to step 47).

Potential solution
Anti-CD3 mAbs combined with anti-CD28 mAbs, IL-2 and IL-15 are strong T-cell activators and stimulate the expansion of CD8 + T cells. However, activation-induced cell death can occur when activating T cells. Activation-induced cell death can be avoided by adjusting mAb and cytokine concentration, duration of the stimulus and seeding density of the T cells. 9 When too many cells die due to activation-induced cell death, keep the cells in culture until sufficient numbers of cells are reached.

Problem 5
Low TCR KO efficiency (related to step 82).

Potential solution
Low TCR KO efficiency can be caused by a poor quality of eSpCas9(1.1)-P2A-EGFP mRNA or the sgRNAs. When electroporating cells with RNA, it is important to keep the RNA on ice and work fast. When the RNA is added to the electroporation cuvette, proceed directly with the electroporation to avoid RNA degradation. Additionally, aliquoting RNAs is important to avoid repeated thawfreezing cycles of the RNAs which can damage and destabilize the RNA. In general, instrument defects could be the cause of poor electroporation efficiencies. In this context, make sure to regularly perform quality controls of the instrument.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Eva Lion (eva.lion@uantwerpen.be).

Materials availability
There are restrictions to the availability of pST1 plasmids generated due to a material transfer agreement for the pST1 plasmid backbone. All information about materials can be addressed to and will be addressed by the lead contact.

Data and code availability
This study did not generate datasets or code.